Clostridium difficile, a spore-forming anaerobic Gram-positive bacillus, is the cause of a spectrum of gastrointestinal illness ranging from mild diarrhe though pseudomembranous colitis, and toxic megacolon. Alarmingly, the incidence, severity and mortality of C. difficile colitis have all increased significantly in the past twenty years. Th mechanism of C. difficile toxicity is well-characterized but no vaccine against C. difficile infecton exists and our knowledge about the interactions of C. difficile with its host during colonization i limited. We have shown that protein subunits of the Type IV pili of C. difficile are associated wit cellular adherence and the initiation of biofilm formation through the association of bacteria into microcolonies. Type IV Pili (T4P) are hair-like surface appendages produced by many species of pathogenic Gram negative bacteria which play a role in diverse processes such as cellular adhesion, colonization, twitching motility, biofilm formation, horizontal gene transfer and in numerous instances are essential for virulence. T4Ps are composed exclusively or primarily of many copies of pilin protein, tightly packed in a helix so that the highly hydrophobic amino-terminus of the pilin is buried in the pilus core. Although better characterized in Gram-negative bacteria, several Gram-positive bacteria, including C. difficile and C. perfringens, have now been shown to produce T4P and T4P genes have been discovered in the genomes of all members of the Clostridium genus. This project aims to characterize the structure, supramolecular assembly and role in biofilm formation of the Type IV pili of C. difficile. Current Research: I have established structural investigations into six putative pilin genes identified in the C. difficile genome and have solved the structure of two, the major pilin subunit PilA1 and a minor pilin, PilJ, by x-ray crystallography. As both of these proteins have been shown to be incorporated into the pilus fiber, I have used these two structures, in combination with data from other sources, to model the structure of a C. difficile Type IV pilus. This model is supported by mutagenesis studies of in vivo pilus assembly performed by a collaborator, Glen Armstrong. Additionally, I have grown isotopically-labeled samples of another pilin, PilA2, and have collected data using NMR spectroscopy allowing me to assign the resonances and calculate the necessary distance constraints to determine the structure by NMR. Initial studies of biofilm formation by C. difficile R202091 and strains with gene-interruptions in pilin genes show clearly that Type IV pili promote biofilm formation in vitro by increasing bacterial self-association. Assays measuring the direct binding of soluble pilin proteins to eukaryotic cells show that a minor pilin, PilJ, directly associates with HeLa, Caco-2 and gastric epithelial cells. The major pilin has a weak affinity for Caco-2 cells while other pilin proteins, including PilW, show no binding. These studies are designed to probe the hypothesis that C. difficile Type IV pili mediate the attachment of in vivo microcolonies to host cells. Independent Phase: My efforts as an independent investigator will be directed towards elucidating the role of C. difficile minor pilins in pilus assembly and biofilm formation. Due to te extreme variability of the major pilin PilA1, when compared to the minor pilins, I hypothesize that any specific protein-protein interactions mediated by Type IV pili involve minor pilins. I propose to test the ability of C. difficile mutants deficient in minor pilins to produce Type IV pili and t form biofilms in vitro. My expectation is that some of these minor pilins will produce fewer or no Type IV pili because they are required for pilus initiation but any that are pilated to wild-type levels but form biofilm at reduced levels are likely involved in specific interactions which mediat bacterial self-association. Investigations into these mechanisms will benefit from continuing investigations into the three-dimensional structures of these proteins, particularly in improving our understanding of how minor pilins are incorporated into the pilus fiber.